U.S. patent number 6,106,806 [Application Number 08/478,733] was granted by the patent office on 2000-08-22 for microbubble-containing contrast agents having a non-proteinaceous crosslinked or polymerised amphiphilic shell.
This patent grant is currently assigned to Nycomed Imaging AS. Invention is credited to Harald Dugstad, Jo Klaveness, Hanno Priebe, P.ang.l Rongved, Roald Skurtveit, Lars Stubberud.
United States Patent |
6,106,806 |
Klaveness , et al. |
August 22, 2000 |
**Please see images for:
( Certificate of Correction ) ** |
Microbubble-containing contrast agents having a non-proteinaceous
crosslinked or polymerised amphiphilic shell
Abstract
Ultrasound contrast agents having microbubbles of gas or a gas
precursor encapsulated by non-proteinaceous crosslinked or
polymerised amphiphilic moieties, e.g. in the form of micelles,
exhibit good stability in vivo upon administration and may if
desired incorporate biodegradable linkages so as to possess
particular desired levels of biodegradability.
Inventors: |
Klaveness; Jo (Oslo,
NO), Priebe; Hanno (Oslo, NO), Rongved;
P.ang.l (Oslo, NO), Stubberud; Lars (Oslo,
NO), Skurtveit; Roald (Oslo, NO), Dugstad;
Harald (Oslo, NO) |
Assignee: |
Nycomed Imaging AS (Oslo,
NO)
|
Family
ID: |
10692381 |
Appl.
No.: |
08/478,733 |
Filed: |
June 7, 1995 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
119217 |
Oct 29, 1993 |
5536490 |
|
|
|
PCTEP9200715 |
Mar 28, 1992 |
|
|
|
|
Foreign Application Priority Data
|
|
|
|
|
Mar 28, 1991 [GB] |
|
|
9106673 |
|
Current U.S.
Class: |
424/9.52 |
Current CPC
Class: |
A61K
49/223 (20130101) |
Current International
Class: |
A61K
49/04 (20060101); A61K 049/04 () |
Field of
Search: |
;424/9.52,9.51,9.5,450,489,493,501,502 ;128/662.02 ;600/458 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0441468 |
|
Aug 1989 |
|
EP |
|
0327490 |
|
Aug 1989 |
|
EP |
|
0327490 |
|
Sep 1989 |
|
EP |
|
0458745 |
|
Nov 1991 |
|
EP |
|
WO-A-9204392 |
|
Mar 1992 |
|
WO |
|
WO-A-9421302 |
|
Sep 1994 |
|
WO |
|
WO-A-9428780 |
|
Dec 1994 |
|
WO |
|
WO-A-9506518 |
|
Mar 1995 |
|
WO |
|
Other References
Juliano et al., STN File Server, File Biosis & Biological
Abstracts, vol. 79, 1985..
|
Primary Examiner: Dees; Jose' G.
Assistant Examiner: Hartley; Michael G.
Attorney, Agent or Firm: Kenyon & Kenyon
Parent Case Text
This application is a continuation-in-part of application Ser. No.
08/119,217 filed on Oct. 29, 1993, U.S. Pat. No. 5,536,490, which
is of 371 of PCT/EP92/00715 dated Mar. 28, 1992.
Claims
We claim:
1. A diagnostic ultrasound contrast agent comprising a stable
dispersion in an aqueous carrier liquid of vesicles comprising
microbubbles of biocompatible gas stabilized at the gas-liquid
interfaces by flexible encapsulating material consisting of
non-proteinaceous cross linked of polymerised amphiphilic
moieties.
2. A contrast agent as claimed in claim 1 wherein said
encapsulating material contains biodegradable linkages selected
from the group consisting of amide, imide, imine, ester, anhydride,
acetal carbamate, carbonate, carbonate ester and disulphide
groups.
3. A contrast agent as claimed in claim 2 wherein said
biodegradable linkages are present in crosslinking groups.
4. A contrast agent as claimed in claim 1 obtained from
polymerisable amphiphilic moieties containing unsaturated
lipophilic chains.
5. A contrast agent as claimed in claim 4 wherein the unsaturated
lipophilic chains are oleyl or linoleyl groups or contain
diacetylene groupings or acryloyl or methacryloyl groupings.
6. A contrast agent as claimed in claim 1 wherein the hydrophilic
portion of the amphiphilic encapsulating material contains one or
more groups selected from quaternary ammonium, hydroxyl, carboxy,
carboxylate ion, amide, phosphate, sulphate and sulphonate.
7. A contrast agent as claimed in claim 6 wherein the hydrophilic
portion of the amphiphilic encapsulating material is the
triglyceryl moiety of a phospholipid, an iodinated X-ray contrast
agent, a carbohydrate, or a choline, ethanolamine, serine or
glycerol residue.
8. A contrast agent as claimed in claim 1 wherein the hydrophilic
portion of the amphiphilic encapsulating material is an optionally
etherified polyoxyethylene glycol residue.
9. A contrast agent as claimed in claim 8 wherein the amphiphilic
encapsulating material is tetraethylene glycol
mono-12-(methacryloyloxy)dodecanoate.
10. A contrast agent as claimed in claim 8 wherein the amphiphilic
encapsulating material is polyethylene glycol (550) methyl ether
12-(methacryloyloxy)dodecanoate.
11. A contrast agent as claimed in claim 8 wherein the amphiphilic
encapsulating material is polyethylene glycol (2000) methyl ether
12-(methacryloyloxy)dodecanoate.
12. A contrast agent as claimed in claim 8 wherein the amphiphilic
encapsulating material is tetraethylene glycol
mono-16-(methacryloyloxy)hexadecanoate.
13. A contrast agent as claimed in claim 8 wherein the amphiphilic
encapsulating material is polyethylene glycol (350) methyl ether
16-(methacryloyloxy)hexadecanoate.
14. A contrast agent as claimed in claim 8 wherein the amphiphilic
encapsulating material is tetraethylene glycol
mono-12-(acryloyloxy)dodecanoate.
15. A contrast agent as claimed in claim 1 wherein the amphiphilic
encapsulating material comprises a membrane-forming lipid and is
crosslinked or polymerised in the hydrophilic portion thereof.
16. A contrast agent as claimed in claim 15 wherein said
membrane-forming lipid comprises at least one phospholipid.
17. A contrast agent as claimed in claim 16 wherein said
membrane-forming lipid comprises a
dialkanoylphosphatidylserine.
18. A contrast agent as claimed in claim 16 wherein said
membrane-forming lipid comprises a blend of an
acylphosphatidylcholine and an acrylphosphatidylserine.
19. A contrast agent as claimed in claim 15 in which the
amphiphilic encapsulating material comprises an oligomer containing
2-20 repeating units.
20. A contrast agent as claimed in claim 1 further containing an
inorganic particulate stabiliser.
21. A contrast agent as claimed in claim 1 which has a half-life in
vivo of 1 to 48 hours.
22. A contrast agent as claimed in claim 21 which has a half-life
in vivo of 1 to 12 hours.
23. An echocardiography contrast agent comprising a stable
dispersion in an aqueous carrier liquid of vesicles comprising
microbubbles of biocompatible gas stabilized at the gas-liquid
interfaces by flexible encapsulating material consisting of
non-proteinaceous cross linked or polymerised amphiphilic moieties,
wherein the microbubbles have an average size of 0.1-10 .mu.m.
24. A method of enhancing ultrasound images of a vascular system
comprising administering to said system a diagnostic ultrasound
contrast agent according to claim 1.
25. A process for the preparation of a contrast agent as claimed in
claim 1 which consists of forming a fluid dispersion of vesicles
comprising gas encapsulated by amphiphilic material and
crosslinking or polymerising said amphiphilic material.
26. A process as claimed in claim 25 wherein the fluid dispersion
is prepared by generating an oil-in-water emulsion in which a
volatile optionally halogenated hydrocarbon is encapsulated by the
amphiphilic material and said volatile hydrocarbon is partially or
completely removed from the vesicles after crosslinking or
polymerisation of the amphiphilic material.
27. A stable aqueous dispersion of vesicles comprising microbubbles
of biocompatible gas stabilized at the gas-water interfaces by
flexible encapsulating material consisting of at least one
non-proteinaceous amphiphilic polymer.
28. A dispersion of vesicles as claimed in claim 27 wherein said
polymer comprising units selected from the group consisting of
lecithins, polyglycerols, polyoxyethylene glycols, polyoxyethylene
glycol ethers, polyoxyethylene derivatized steroids, glycosides,
galactosides, hydroxyacids, polyhydroxyacids, carbohydrates,
aminoalcohols, cyanoacrylates, acrylamides, and hydroxyamides.
29. A dispersion of vesicles as claimed in claim 27 wherein the
hydrophilic portion of said polymer comprises an optionally
etherified poloxyethylene glycol residue.
30. A physiologically acceptable dispersion of vesicles according
to claim 27.
31. A diagnostic ultrasound contrast agent comprising a dispersion
in an aqueous carrier liquid of microbubbles of biocompatible gas
stabilized at the gas-liquid interfaces by flexible encapsulating
material comprising non-proteinaceous crosslinked or polymerized
amphiphilic moieties containing biodegradable linkages wherein the
biodegradable crosslinking groups include units of formula
where Y and Z, which may be the same or different, are --O--, --S--
or --NR.sup.3 --; R.sup.1 and R.sup.2, which may be the same or
different, are hydrogen atoms or carbon-attached monovalent organic
groups or together represent a carbon-attached divalent organic
group, R.sup.3 is a hydrogen atom or an organic group, and the
symbols n, which may be the same or different, are zero or 1.
Description
This invention relates to novel contrast agents, more particularly
to new gas-containing or gas-generating contrast agents of use in
diagnostic ultrasonic imaging.
It is well known that ultrasonic imaging comprises a potentially
valuable
diagnostic tool, for example in studies of the vascular system,
particularly in cardiography, and of tissue microvasculature. A
variety of contrast agents has been proposed to enhance the
acoustic images so obtained, including suspensions of solid
particles, emulsified liquid droplets, gas bubbles and encapsulated
gases or liquids. It is generally accepted that low density
contrast agents which are easily compressible are particularly
efficient in terms of the acoustic backscatter they generate, and
considerable interest has therefore been shown in the preparation
of gas-containing and gas-generating systems.
Initial studies involving free gas bubbles generated in vivo by
intracardiac injection of physiologically acceptable substances
have demonstrated the potential efficiency of such bubbles as
contrast agents in echocardiography; such techniques are severely
limited in practice, however, by the short lifetime of the free
bubbles. Interest has accordingly been shown in methods of
stabilising gas bubbles for echocardiography and other ultrasonic
studies, for example using emulsifiers, oils, thickeners or
sugars.
WO 80/02365 discloses the use of gelatin-encapsulated gas
microbubbles for enhancing ultrasonic images. Such microbubbles do
not, however, exhibit adequate stability at the dimensions
preferred for use in echocardiography (1-10 .mu.m) in view of the
extreme thinness of the encapsulating coating.
U.S. Pat. No. 4,774,958 discloses the use of microbubble
dispersions stabilised by encapsulation in denatured protein, e.g.
human serum albumin. Such systems permit the production of
microbubble systems having a size of e.g. 2-5 .mu.m but still do
not permit efficient visualisation of the left heart and
myocardium.
EP-A-0327490 discloses, inter alia, ultrasonic contrast agents
comprising a microparticulate synthetic biodegradable polymer (e.g.
a polyester of a hydroxy carbonic acid, a polyalkyl cyanoacrylate,
a polyamino acid, a polyamide, a polyacrylated saccharide or a
polyorthoester) containing a gas or volatile fluid (i.e. having a
boiling point below 60.degree. C.) in free or bonded form.
Emulsifiers may be employed as stabilisers in the preparation of
such agents, but such emulsifiers do not chemically interact with
the polymer.
We have now found that particularly effective ultrasonic contrast
agents may be obtained by encapsulating gas bubbles or gas
generating systems with polymers containing chemically linked
surface active, i.e. amphiphilic, moieties. Thus the surface active
properties of the amphiphilic groups stabilise the microbubble
system by reducing surface tension at the gas-liquid interfaces,
e.g. by forming monolayers or one or more bilayers (alternatively
known by the terms micelles, vesicles, liposomes and niosomes) at
said interfaces, while the linking of the groups through the
polymer system generates further stability. Flexibility of the
encapsulating materials also enhances the image density afforded by
such contrast agents. For simplicity the terms "vesicle" is used
herein to denote all such microbubble structures prior to or after
crosslinking or polymerisation. It should be noted that under some
conditions irregularly shaped structures may be formed, e.g.
microtubles which may join with or even entrap spherical
structures.
Thus according to one aspect of the present invention there are
provided contrast agents for use in diagnostic ultrasound studies
comprising microbubbles of gas or a gas precursor encapsulated by
non-proteinaceous crosslinked or polymerised amphiphilic
moieties.
The term "crosslinked" is used herein to denote that the
amphiphilic moieties are chemically linked to each other, e.g. to
form a polymeric structure which may incorporate one or more
polymer systems (including copolymers), and includes systems
prepared by reaction with so-called zero crosslinking agents. The
terms "polymerised" and "polymeric structure" as used herein
embrace low molecular weight polymer systems such as dimers and
other oligomers.
A major advantage of contrast agents according to the invention is
that they may be designed to a particular desired level of
biodegradability in vivo by selecting appropriate biodegradable
linkages at appropriate positions. It will be appreciated that in
order to be effective the contrast agents must be stable throughout
the ultrasonic examination but are preferably metabolised or
removed safely from the circulation system shortly thereafter.
Contrast agents in accordance with the invention should thus
preferably have a half-life in vivo of not more than 48 hours, for
example 1-12 hours.
Biodegradable linkages which may be present in contrast agents
according to the invention include amide, imide, imine, ester,
anhydride, acetal, carbamate, carbonate, carbonate ester and
disulphide groups. At least one such group should preferably be
present in the amphiphilic moiety, in the hydrophilic and/or
lipophilic portion, it may be advantageous to position the group in
the hydrophilic part to facilitate enzymic interaction in vivo. It
is further preferred that biodegradable linkages be present in the
polymer backbone to ensure substantial breakdown of the polymer in
the body.
Any biocompatible gas may be employed in the contrast agents of the
invention, for example air, nitrogen, oxygen, hydrogen, nitrous
oxide, carbon dioxide, helium, argon, sulphur hexafluoride, low
molecular weight optionally fluorinated hydrocarbons such as
methane, acetylene, carbon tetrafluoride and other perfluoroalkanes
such as perfluoropropane, perfluorobutane and perfluoropentane, and
mixture of any of the foregoing. The term "gas" as used herein
includes any substances, including mixtures, in gaseous or vapour
form at 37.degree. C. In general the gas may be free within the
microbubbles, advantageously in the form of a gas-filled
"microballoon" since the echogenicity of such products may be
enhanced by virtue of their relatively flexible nature.
Alternatively the gas may be trapped or entrained within a
containing substance.
Gas precursors include carbonates and bicarbonates, c.g. sodium or
ammonium bicarbonate and aminomalonate esters. The term "gas
precursor" as used herein also embraces substances such as volatile
hydrocarbons which may initially be encapsulated but thereafter are
partially or completely removed from the vesicles, e.g. by
evaporation or freeze-drying, to be replaced by gas.
For applications in echocardiography, in order to permit free
passage through the pulmonary system and to achieve resonance with
the preferred imaging frequency of about 0.1-15 MHz, it may be
convenient to employ microbubbles having an average size of 0.1-10
.mu.m, e.g. 1-.mu.m. Substantially larger bubbles, e.g. with
average sizes of up to 500 .mu.m, may however be useful in other
applications, for example gastrointestinal imaging or
investigations of the uterus or Fallopian tubes.
If desired the microbubbles may incorporate particulate
stabilisers, for example inorganic materials such as silica or iron
oxide which are only partially wetted by the solvent system
employed, e.g. having a particle size of 1-500 nm. Colloidal silica
having a particle size of 5-50 nm may advantageously be employed
for this purpose.
Polymer systems which may be employed in the contrast agents of the
invention include carbohydrates such as dextrans and starches,
chitin, chitosan, carboxymethylchitosan, aliginate, hyaluronic
acid, polyacrylamides, polycyanoacrylates,
hydroxyalkylpolycyanoacrylates, polyhydroxy acids such as
polylactic acids, polyhydroxybutyrates, polyglycolic acids,
polylactide-glycolides, polyorthoesters, polyanhydrides,
polyurethanes, polyester imides, polyimides, polyacetals,
poly-epsilon-caprolactones, polydioxanones, polyaminotriazoles,
poly(amide-enamines), poly(amide-urethanes), polyphosphazenes,
polyvinyl alcohols, organo-polysiloxanes, poly(enol-ketones) and
copolymers of these materials, modified as necessary to introduce
hydrophilic or lipophilic moieties.
The contrast agents according to the invention may, for example, be
prepared by forming a fluid (e.g. aqueous) dispersion of vesicles
comprising a gas or gas precursor encapsulated by amphiphilic
material followed by crosslinking or polymerisation of the
amphiphilic material. Alternatively crosslinking or polymerisation
may be effected prior to or during vesicle formation.
The vesicles will normally comprise a substantially spherical
monolayer or multilayer of the amphiphilic material. The
hydrophilic moieties of the amphiphiles will be physically
associated to form a contiguous layer while the lipophilic moieties
will also form a layer which may be inside or outside the
hydrophilic layer. In bilayers, two layers of the amphiphilic
material may be superimposed; thus, for example, a first layer of
amphiphilic material may form in which the lipophilic groups are on
the outside. A second layer of amphiphilic material may then
overlay the first layer with the lipophilic groups adjacent to the
lipophilic groups of the first layer and the hydrophilic groups on
the outside. Similarly, a bilayer may have the lipophilic groups on
the outside and inside and the hydrophilic groups sandwiched
between.
Where the fluid in which the vesicles are dispersed is polar, for
example aqueous, the hydrophilic groups of the vesicles will tend
to be on the outside of the micelles and the lipophilic groups will
be on the inside forming a monolayer. On the other hand, if the
dispersing fluid is apolar, the lipophilic groups will be on the
outside, particularly if the encapsulated material is hydrophilic,
e.g. a gas precursor or a solid material containing absorbed or
entrained gas, possible in association with a polar liquid.
Bilayers may form when the encapsulated material is of the same
type, i.e. hydrophilic or lipophilic, as the dispersing fluid.
The amphiphiles used in accordance with the present invention will
carry functional groups permitting crosslinking or polymerisation.
These may in some instances be groups imparting hydrophilic or
lipophilic character or they may be independent of the amphiphilic
groupings.
The amphipiles may be considered in three categories:
1. The amphiphiles may carry at least two simple reactive groups
such as hydroxyl, amino or carboxyl groups which are capable of
reacting with polyvalent reactive monomers, such as crosslinking
agents, or preformed polymers. For example, if the amphiphile
carries two hydroxyl groups (in the hydrophilic moiety), a
dicarboxylic acid such as suberic acid may be reacted with the
vesicles after encapsulation of the gas or gas precursor to provide
a crosslinked or polymerised structure. Diamino-amphiphiles may
similarly be reacted with dicarboxylic acids or dialdehydes such as
glutaraldehyde, while dicarboxylic amphiphiles may be reacted with
diamines or diols. Amphiphiles containing both free amino and
carboxyl groups may be polymerised to form amide polymers by
reaction with a carboxyl group activator such as a carbodiimide,
advantageously a water-soluble carbodiimide such as
N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride; such
materials may also be regarded as zero crosslinking agents.
Additional crosslinking may be provided by trifunctional reagents.
A catalyst may be employed to assist reaction.
The crosslinking agent may itself be amphiphilic so that the
vesicle will form with the lipophilic and hydrophilic groups of the
first amphiphile and the amphiphilic crosslinking agent in
alignment, whereupon crosslinking between the reactive functional
groups may be initiated.
As indicated above, it is particularly advantageous for the
polymerised or crosslinked amphiphile to be biodegradable,
especially into relatively simple water soluble units. In the case
of the ester and amide bonds referred to above, esterase and
amidase enzymes will commonly be available in the vascular system
and can degrade the encapsulating material back to separate
amphiphile molecules and the diamine, diol or diacid reagents which
under physiological conditions will not recombine.
If desired, even more biolabile crosslinking groups such as
carbonate ester groups may be introduced e.g. using orthoester
crosslinking agents. Another useful class of crosslinking agents
have the formula (I)
(where Y and Z, which may be the same or different, are --O--,
--S-- or --NR.sup.3 --;
R.sup.1 and R.sup.2, which may be the same or different, are
hydrogen atoms or carbon-attached monovalent organic groups or
together represent a carbon-attached divalent organic group;
R.sup.3 is a hydrogen atom or an organic group;
the symbols n, which may be the same or different, are zero or
1;
R.sup.8 and R.sup.9, which may be the same of different are
divalent organic groups, for example alkylene or alkylidene groups
having 1-12 carbon atoms; and
A.sup.1 and A.sup.2 are functional groups, for example reactive
with hydroxyl, amino or carboxyl groups), since the crosslinking
groups so introduced contain units of formula
(where Y, Z, each n, R.sup.1 and R.sup.2 are as defined above)
which are particularly readily degraded by common esterases, while
exhibiting stability in the absence of enzymes.
R.sup.1, R.sup.2 and R.sup.3 may each be a hydrocarbyl or
heterocyclic group, for example having 1-20 carbon atoms, e.g. an
alkyl or alkenyl group (preferably having up to 10 carbon atoms), a
cycloalkyl group (preferably having up to 10 carbon atoms), an
aralkyl group (preferably having up to 20 carbon atoms), an acyl
group (preferably having up to 20 carbon atoms) or a heterocyclic
group having up to 20 carbon atoms and one or more heteroatoms
selected from O, S and N; such a hydrocarbyl or heterocyclic
grouping may carry one or more functional groups such as halogen
atoms or groups of the formulae --NR.sup.4 R.sup.5, --CONR.sup.4
R.sup.5, --OR.sup.6, --SR.sup.6 and --COOR.sup.7, where R.sup.4 and
R.sup.5, which may be the same or different, are hydrogen atoms,
acyl groups or hydrocarbyl groups as defined for R.sup.1 and
R.sup.2 ; R.sup.5 is a hydrogen atom or an acyl group or a group as
defined for R.sup.1 or R.sup.2 and R.sup.7 is a hydrogen atom or a
group as defined for R.sup.1 or R.sup.3 ; where R.sup.1 and R.sup.2
represent a divalent grouping, this may for example be an alkylene
or alkenylene group (preferably having up to 10 carbon atoms) which
may carry one or more functional groups as defined above. In
general R.sup.1 and R.sup.2 are preferably hydrogen or small groups
such as C.sub.1-4 alkyl groups.
2. The amphiphile may contain polymerisable groupings which can be
caused to polymerise after vesicle formation. Such polymerisable
groupings may, for example, include unsaturated lipophilic chains,
e.g. alkenyl or alkynyl groupings containing up to 50 carbon atoms,
for example 10-30 carbon atoms, such as oleyl or linoleyl groups or
groups containing diacetylene, acryloyl or methacryloyl groupings.
Polymerisation of such groupings will, in general, yield
hydrocarbon backbone polymers the backbones of which are not
readily biodegradable, although such polymers may be designed so
that the backbone residue resulting from biodegradation is
water-soluble, e.g. by virtue of the presence of hydrophilic
substituents such as carboxyl or hydroxyl groups, to enhance its
dispersibility. The chain length of such polymers is in general
preferably such that their molecular weight does not exceed
40,000.
Where a greater degree of biodegradability is required, it may be
preferable to avoid formation of polymeric hydrocarbon chains which
cannot readily be degraded and to effect polymerisation or
crosslinking exclusively through biodegradable groups such as
ester, carbonate, carbamate, amide or imide bonds of the type
referred to above. In general, the functional groups leading to
such bonds will be hydrophilic and thus lead to crosslinking
between the hydrophilic parts of the amphiphiles.
However, polymerisation of lipophilic hydrocarbon chains may be
used to yield a biodegradable polymer if the amphiphile comprises a
biodegradable hydrophilic moiety carrying two such chains; where
the lipophilic chains of adjacent amphiphile molecules become
crosslinked, e.g. via unsaturated carbon-carbon bonds, the extended
lipophilic groupings so formed will be separated by the
biodegradable hydrophilic groups; on biodegradation, the polymeric
structure will thus break up into relatively small lipophilic
molecules carrying the residues of the degraded hydrophilic
moieties.
3. A soluble amphiphilic polymer carrying appropriate functional
groups may be further polymerised or crosslinked after vesicle
formation. Such substances include polyamino acids and
carbohydrates carring lipophilic
groups, as well as low molecular weight polyesters, polyamides etc
carrying appropriate groups providing amphiphilic character. Thus,
for example, hydrophilic polymers, such as those listed above, may
be provided with lipophilic chains, e.g. C.sub.10-30 alkyl, alkenyl
or alkynyl groups, to provide suitable amphiphiles for use in
accordance with the invention. Chemical methods for the attachment
of such lipophilic chains include partial esterification of the
hydroxyl groups of polyhydroxy acids, salt formation of anionic
surfactants on the amino groups of chitosan or covalent
derivatisation of such groups, and attachment of hydrophobic groups
to carbohydrates or cyclodextrins by way of ester bonds.
The soluble polymer for further polymerisation may also be an
amphiphile polymerised or crosslinked in accordance with (1) or (2)
above.
Polymerisable or crosslinkable amphiphiles which may be used in
accordance with the invention thus include compounds of the general
formula (II).
where X is an anionic, cationic or non-ionic hydrophilic
moiety;
R.sup.10 is a lipophilic group;
B is a group capable of polymerisation or crosslinking;
p and q are integers; and
r is zero or, when neither X or R.sup.10 is capable of
polymerisation or crosslinking, is an interger.
The groups X and R.sup.10 may be joined in various ways. Thus, for
example, a hydrophilic group X may carry one or several lipophilic
groups R.sup.10 or a lipophilic group R.sup.10 may carry one or
several hydrophilic groups X. One or more hydrophilic groups X may
also join separate lipophilic groups R.sup.10 as long as the
amphiphile can adopt a configuration in which the hydrophilic and
lipophilic moieties of adjacent molecules are aligned.
Similarly, the group(s) B (where present) may be attached to one or
more of the groups X and R.sup.10.
To provide or enhance biodegradability, one or more biodegradable
groupings W may connect the groups X, R.sup.10 and B.
The group X may, for example, be a quaternary ammonium grouping
--N(R.sup.11).sub.3 V where the groups R.sup.11 (which may be the
same or different) may be, for example, alkyl, aralkyl or aryl
groups containing, for example, up to 20 carbon atoms, and V is an
anion. It will be appreciated that one or more of the groups
R.sup.22 may be a lipophilic group R.sup.10.
Other useful hydrophilic groups X include, hydroxyl, carboxylate,
amide, phosphate, sulphate and sulphonate groups. Further examples
of hydrophilic groups X include:
--O.CH.sub.2.CH.sub.2.N.sup.+ (CH.sub.3).sub.3 (choline)
--O.CH.sub.2.CH.sub.2.N.sup.+ H.sub.3 (ethanolamine)
--O.CH(NH.sub.3.).COO.sup.- (serine)
--O.CH.sub.3.CH(OH).CH.sub.2 OH (glycerol)
--hexoses and pentoses such as inositol.
The group R.sup.10 may, for example, be a saturated or unsaturated,
straight or branched hydrocarbon chain, which may contain, for
example, 6-50 carbon atoms and may be interrupted by one or more
biodegradable groups W and may carry one or more functional groups
permitting chains R.sup.10 on adjacent amphiphiles to crosslink to
form a biodegradable group. Useful groups R.sup.10 include oleyl
and linoleyl groups and chains containing diacetylene
groupings.
The group(s) B may be, for example, orthoester groups which form
carbonate ester linkages with hydroxyl groups, or hydroxyacid
groups (or separate hydroxyl and carboxyl groups) which form ester
linkages.
It will be appreciated that the hydrophilic group X may comprise a
moiety which is not itself directly responsible for hydrophilic
properties, as in the case of a group R.sup.11 of a quaternary
ammonium groupings as defined above, which may for example be a
lower alkyl group too small to impart lipophilic character; such
groups may also form part of the connection between the groups X
and R.sup.10. In other words, there may be transitional regions
between groups X and R.sup.10 which are not strictly either
lipophilic or hydrophilic in themselves but can be regarded as part
of either X or R.sup.10.
Thus, in a special case of the amphiphiles of formula (II), the
groups X, R.sup.10 and B may be attached to a preformed polymer
which may be regarded as part of X or of R.sup.10 according to its
chemical and physical character. Such a polymer may be a known
hydrophilic polymer on to which lipophilic groups (as discussed
above) have been attached, or a lipophilic polymer, e.g. a
polyolefin, carrying hydrophilic groups. Alternatively, such a
polymer may be obtained by partial polymerisation of an amphiphile
of formula (II). In all such cases, the preformed polymer should be
sufficiently soluble to permit vesicle formation and should be so
functionalised as to permit covalent, ionic or coordinate
crosslinking to stablise the vesicles.
Particularly useful monomeric amphiphiles include cyanoacrylate
esters carrying lipophilic esterifying groups (which may also have
hydrophilic moieties). Thus, for example, U.S. Pat. No. 4,329,332
describes the micellar polymerisation of lower alkyl
cyanoacrylates, a technique which may be extendable to the
polymerisation of acrylates of the formula CH.sub.2
.dbd.C(CN).CO.O.(C.sub.6-20 aliphatic). Similarly, a di-acrylate of
the formula
has been used by Ping et al (Int. J. Pharm, 61 (1990) 79-84).
Corresponding cyanoacrylates may also be used.
Amphiphilic materials of use in accordance with the invention
include the following classes of substances derivatised with
lipophilic groups:
lecithin derivatives,
polyglycerol,
polyoxyethylene glycol and ethers thereof,
polyoxyethylene derivatives of steroids,
glycosides,
galactosides,
hydroxyacids or polyhydroxyacids (including carboxylic, phosphonic,
sulphonic and sulphinic acids),
carbohydrates and derivatives thereof,
aminoalcohols and derivatives thereof,
cyanoacrylates,
acrylamides, and
hydroxyamides.
Crosslinkable and Polymerisable Amphiphiles
A number of classes of useful crosslinkable and/or polymerisable
amphiphiles are listed below:
1. CH.sub.2
(OB.sub.1).CH(OB.sub.2).CH.sub.2.O.PO(O.sup.-)O(CH.sub.2).sub.2
N.sup.+ (CH.sub.3).sub.3
where B.sub.1 and B.sub.2 may be
Such compounds may thus be prepared by procedures described in
EP-A-0032622. The zwitterionic group may be introduced by
subjecting the appropriate phosphonic or phosphinic acid or an
esterifiable derivative thereof to reaction with glycerol or an
esterifiable derivative thereof. The groups B.sub.1 and B.sub.2 may
be introduced into the molecule by esterification using the
carboxylic acid of B.sub.1 and B.sub.2 or an ester-forming
derivative thereof. These reactions can be carried out between the
glycerol or derivatives thereof on the one hand, and the carboxylic
acid and the phosphorus ester on the other, either simultaneously
or optionally in steps. Other known methods for the synthesis may
equally well be used.
Polymerisation of these compounds may, for example, be obtained by
irradiation at 254 nm using a xenon lamp after formation of gas
containing liposomes or formation of monolayers of the amphiphiles
at the gas/liquid interface.
2. Phospholipids such as phosphodiglycerides and sphingolipids
carrying polymerisable groups.
3. Unsaturated oils having hydrophilic groups such as corn oil,
sunflower seed oil, soybean oil, safflower oil, peanut oil,
cottonseed oil and olive oil.
4. Saturated and unsaturated fatty acid derivatives with hydroxyl
groups, for example castor oil and ergot oil which are
triglycerides of d-12-hydroxyoleic acid.
5. Compounds as described in "Polymerised Liposomes" (Technical
Insights Inc 1988) and Hub et al (J. Macromol.Sci.Chem. A15, (5),
1981, 701-715). These may have the structures: ##STR1##
6. Compounds of the formula:
where L and M may be --O--, --S-- or --NR.sup.12 -- (where R.sup.12
is H or an alkyl group), for example the compounds in which
L.dbd.M.dbd.--O--; L.dbd.--O--, M.dbd.--N(CH.sub.3)--;
L.dbd.--NH--, M.dbd.O ;
L.dbd.--O--, M.dbd.--N.sup.+ (CH.sub.3).sub.2 -- Br and
L.dbd.--O--, M.dbd.--N(CH.sub.2.CH.sub.2.SO.sub.3 H)--
Such compounds may be prepared by reacting a reactive derivative of
hexacosane-10,12-diynoic acid (e.g. the acid chloride) with the
appropriate compound (HLCH.sub.2 CH.sub.2).sub.2 M in dry
chloroform at 0.degree. C. in the presence of pyridine, if
necessary followed by quaternisation.
Synthesis of hexacosane-10,12-diynoic acid is described by Singh et
al (polym.Prep.: Am.Chem.Soc. Div.Polym.Chem; 26 (2), 1985, 184-5).
The acid chloride may be prepared by reaction with
oxalylchloride.
7. Compounds as described by Paleos (Chem.Soc.Rev. 14, 1985,
45-67), for example of the following structures: ##STR2##
8. Esters of .alpha.-amino fatty acids which may be self condensed
as described by Folda et al (Rapid.Commun. 3, 1982, 167-174) e.g.
methyl 2-aminooctadecanoate, docosanyl 2-aminooctadecanoate, methyl
2-aminohexcosanoate and docosanyl 2-amino-hexacosanoate.
These esters of the long chain amino acids may be synthesized from
the saturated carboxylic acids by .alpha.-bromination using the
Hell-Volhard-Zelinsky reaction. The resulting .alpha.-bromo acids
are converted to the corresponding amino acid by the method of
Cheronis et al (J.Org.Chem. 6 (1949) 349). The methyl esters of the
amino acid hydrochlorides are prepared by passing dry HCl-gas
through a suspension of the amino acid in refluxing methanol. The
docosanyl ester of the amino acid hydrochlorides are synthesized by
passing dry HCl-gas through a 1:1 mixture of amino acid and
docosanol at 110.degree. C. The ester hydrochlorides are then
suspended in dry chloroform and converted to the free amine by
passing dry ammonia through the suspension.
9. Long chain esters of sulphosuccinic acid carrying polymerisable
functions.
10. Long chain esters of pyridinum dicarboxylic acids (e.g.
3,5-dicarboxy 1-methyl pyridinum iodide) carrying polymerisable
functions.
11. Iodinated X-ray contrast agents carrying long chain ether or
ester groups having polymerisable functions. Thus, for example, an
X-ray contrast agent derived from iothalamic acid may have multiple
N-dihydroxyalkyl groups one or two of which may be esterified with
long chain fatty acids. Thus, for example, iohexol may be partially
protected by forming an acetonide derivative of two of the three
dehydroxy alkyl groups, followed by reaction with an activated
fatty acid, e.g. the acid chloride, and deprotection to remove the
acetonide groups. Such an amphiphile may readily be cross-linked by
reaction with a dicarboxylic acid after vesicle formation.
12. Di-fatty acid esters of sorbitan. The multiple free hydroxyl
groups which are present permit cross-linking by diacids.
Alternatively, the esterifying fatty acid groups may be unsaturated
to permit olefinic addition polymerisation.
13. Diesters of the formula
where R.sup.14 is a hydrophilic group and each R.sup.13 is a
lipophilic group, at least one of R.sup.13 and R.sup.14 carrying a
polymerisable group and/or functional groups permitting
crosslinking. Such compounds may be synthesised by reaction of a
dihalide of the formula R.sup.14.CH.Hal.sub.2 with a salt of an
acid R.sup.13.COOH. They are particularly readily
biodegradable.
It may also be beneficial to include in the encapsulating material
one or more further amphiphiles such as cholesterol which are not
bonded or polymerised but serve to improve the stability and/or
flexibility of the microbubbles.
14. Crosslinkable and/or polymerisable membrane forming lipids,
preferably which are capable of crosslinking or polymerisation in
the hydrophilic portions thereof.
As is recognised in the surfactant art, membrane-forming lipids may
have the characteristic that they form liquid crystalline bilayers
in aqueous media. Typically their molecular geometry is such that
the hydrophilic and hydrophobic portions are of comparable size.
Membrane-forming lipids also include amphiphiles which form
monolayers or single bilayers at gas-water interfaces (e.g. as in
Langmuir-Blodget films). Preferred membrane-forming lipids include
lipids such as are found in biological membranes which are
characterised by low water solubility and a tendency in aqueous
solutions substantially to decrease surface tension, e.g. to almost
zero; such lipids typically form liquid crystalline bilayers at low
concentration in aqueous media.
One preferred class of membrane-forming lipids useful in this
embodiment of the invention comprises phospholipids such as
diacylphosphatidylcholines and diacylphosphatidylserines,
particularly dialkanoyl phosphatidylserines such as dipaimitoyl and
distearoyl phosphatidylserines. Corresponding
diacylphosphatidylglycerols, phosphatidylethanolamines and
phosphatidydlinositols are also advantageous. Other lipids which
may be used include mono- and di-glyceride esters of fatty acids,
sphingolipids, glycolipids, glycerolipds and carbohydrate esters of
fatty acids. It will be appreciated that at least one
membrane-forming lipid should desirably contain functional groups
in the hydrophilic portion thereof which are capable of reaction
under appropriate conditions and/or with appropriate reagents to
permit crosslinking and/or polymerisation as required,
advantageously so as to generate biodegradable linkages, for
example comprising amide, imide, imine, ester, anhydride, acetal,
carbamate, carbonate, carbonate ester or disulphide groups.
By virtue of the high degree of stability which may be imparted by
membrane-forming lipids it may be possible to incorporate other
components, e.g. surfactants or cosurfactants, into the stablishing
crosslinked or polymerised membrane-forming lipid material, even to
the extent that the crosslinked or polymerised membrane-forming
lipid represents a minor component of the encapsulating material,
while maintaining adequate product stability. Thus the
encapsulating membrane material may incorporate other components,
for example to modify membrane properties such as stability,
dispersibility, aggregation tendency, biological activity,
flexibility or polarity. Representative additives include sterols
such as cholesterol and non-crosslinkable and non-polymerisable
phospholipids. Alternatively the membrane-forming lipid may be
subjected to a relatively low degree of crosslinking or
polymerisation so that the encapsulating membrane material of the
contrast agent product includes a proportion of unreacted (e.g.
monomeric) membrane-forming lipid.
The product may comprise a blend of membrane-forming lipids, e.g.
such that the membrane-forming properties are superior to those of
the individual components. Blends of membrane-forming lipids may,
for example, include mixtures of an acylphosphatidylcholine and an
acylphosphatidylserine.
Other components of such blends may include sterols such as
cholesterol and/or membrane components carrying surface-modifying
groups such as polyethylene glycol moieties.
As a result of the aforementioned high degree of stability which
may be imparted by membrane-forming lipids it may be possible to
employ a low degree of crosslinking or polymerisation in such
contrast agents, for example to enhance the flexibility of the
encapsulating material, which in turn will enhance the image
density afforded by the contrast agents. Oligomers, e.g. containing
2-20 repeating units, are one preferred category of
membrane-forming lipids in accordance with this embodiment of the
invention.
As indicated above the microbubbles may in general be stabilised by
incorporation of particulate material together with encapsulated
gas. Such particles include, for example, silica and iron oxide.
The preferred particle size for such stabilising particles is in
the range 1 to 500 nm, depending on the size of the microbubbles.
The particles should be such that they are only partially wetted by
the fluid medium used to disperse the micelles, i.e. the contact
angle between the material of the particles and the fluid should be
about 90 degrees.
The stabilising particles may carry functional groups which will
interact with the amphiphiles to form covalent or other linkages.
Particles of the polymerised amphiphiles of formula (II) may be
useful in this context. Colloidal silica particles may have a
particle size in the range 5-40 nm and may carry silanol groups on
the surface which are capable of interaction with the amphiphile by
hydrogen bonding or by forming covalent bonds.
The amphiphile by hydrogen bonding or by forming covalent
bonds.
The amphiphiles may stabilize the gas or gas precursor by forming a
monolayer at the interface between the liquid medium and the gas or
gas precursor system, or by forming vesicles consisting of one or
more layers containing the gas or gas precursor. The liquid medium
may be water or an any non-aqueous liquid with polar, protic,
aprotic or apolar characteristics.
The stabilisation of the system by monolayers or multilayers or the
formation of the vesicles may be activated, as fully described in
the literature, by sonication or shaking of the amphiphilic
material mixture in an appropriate medium, e.g. in the presence of
an appropriate gas, or the vesicles may be formed by any
conventional liposome/vesicle-forming principle.
The amphiphiles may form conventional micelles, or inverse micelles
when using an apolar non-aqueous medium. The stabilized systems may
be dried or freeze-dried or the non-aqueous phase may be
evaporated. Such dried systems may be stored substantially
indefinitely, e.g. under an atmosphere of a gas or gas mixture
which is desired to be incorporated into the product, and may
subsequently be resuspended in any physiological acceptable solvent
such a saline or phosphate buffer, optionally using a suspending or
emulsifying agent.
The methods of polymerization used for the stabilisation of the
vesicles, are well established methods in polymer chemistry, i.e.
as described in "Comprehensive Polymer Science", Vol 1-7, Pergamon
Press, Oxford 1989, or "Methoden der Organischen Chemie",
Houben-Weyl, Makromolekulare Stoffe Band E20/1-3, Georg Thieme
Verlag, Stuttgart 1987. Examples of suitable methods may be chain
polymerization methods such as ionic or radical polymerisation or
metal catalysed polymerisation, or the systems may polymerize
spontaneously by step polymerisation when monolayers or vesicles
are formed. Initiators may be UV-irradiation or simple pH-change,
or radial initiators. Particularly interesting here may be
encapsulation of a substance which, by slight increase in
temperature develops a gas, and simultaneously generates free
radicals which initiates polymerisation of the surrounding shell.
Such a substance is described in "Comprehensive Polymer Science",
Vol 3, Pergamon Press, Oxford 1989, p.p. 99, i.e.
azo-bis-isobutyronitrile (AIBN), which by UV-irradiation, or by
warming to 40.degree. C. starts generating N.sub.2 while generating
two molecules of cyano-isopropyl radicals which may initiate
polymerisation or rapidly pair. Polymerisation of amphiphiles
containing unsaturated groupings may also be initiated by
sonication (see Price et al., Brit. Polym. J. 23 (1990), 63-66),
e.g. when this is used to generate a gas-in-liquid emulsion as
described in greater detail hereafter.
A gas entrapped system may be obtained by using a gas precursor or
the gas itself may be entrapped. The gas may be entrapped into the
amphiphile mixture simply by vigorously shaking the mixture in the
presence of air, i.e. creating a gas-in-liquid emulsions a s
described in U.S. Pat. No. 4,684,479. Another well established
method, described e.g. in U.S. Pat. No. 4,774,958 for creating a
gas containing bubble is by sonication of the mixture in the
presence of air. Another well known method comprises passing pas
through a syringe into a mixture of amphiphile and liquid. As
described in U.S. Pat. No. 3,900,420 the microgas-emulsion may be
created by using an apparatus for introducing gas rapidly into a
fast-flowing liquid. A region of low pressure is created in a
liquid containing the amphiphile. The gas is then introduced to the
region of low pressure and the gas-in-liquid system is obtained by
pumping the liquid through the system.
By using the principle of electrolysis it is possible to generate
the gas to be entrapped directly in a container containing the
amphiphiles. The electrolytes necessary for the electrolysis may
even help to further stabilize the amphiphiles to make the
polymerisation possible. An aqueous solution containing
electrolytes may generate hydrogen gas at the cathode and oxygen at
the anode. The electrodes may be separated by a salt bridge. On
adding hydrazine nitrogen gas may be generated at the anode. Using
the Kolbe reaction, one may also generate CO.sub.2 from carboxylic
acids using electrolysis.
As described above, gas entrapped vesicles may be obtained by
forming liposomes or vesicles consisting of one or more bilayers.
These vesicles may be formed at elevated pressure conditions in
such a way that the gas is entrapped in the vesciles. In general it
may be advantageous during processing to employ a perfluoroalkane
or other gas with similar low water solubility to ensure adequate
persistence of the microbubbles during the time taken for formation
of the stabilising encapsulating vesicles and their crosslinking or
polymerisation.
It is also possible to form a liquid-liquid (e.g. oil-in-water
emulsion in the presence of amphiphile systems as discussed above,
e.g. by sonication or shaking, to form liquid-containing vesicles
which may be polymerised prior to, during or after vesicle
formation. The polymerised vesicles may then be treated to remove
the liquid (conveniently a volatile water-immiscible hydrocarbon,
e.g. a halogenated hydrocarbon such as a freon) therefrom by
evaporation, where the boiling point of the liquid is relatively
low, or by extracting with a low-boiling solvent which can itself
be removed by evaporation. Evaporation of low-boiling liquid cores
may also occur spontaneously during sonication. Where the liquid in
the vesicles is water, it can be removed by freeze drying. In
general such dried products may be stored under an atmosphere of a
gas or gas mixture which is desired to be incorporated into the
final product.
The following Examples are given by way of illustration only.
Bis-linoleyl-lecithin is commercially available from Lipids
Products, Surrey, UK.
EXAMPLE 1
A saturated solution of the bis-linoleyl-lecithin in an aqueous
medium is obtained by mixing 100 mg of the amphiphile in 100 ml of
sterile, pyrogen free water. The saturated solution is filtered
through a 0.45 .mu.m filter, and the resulting solution is
sonicated for 1-10 minutes in the presence of air. During the
sonication, air is entrapped into the solution and a gas-in-liquid
emulsion is formed. Polymerization of the monolayer of the
amphiphiole at the gas-liquid interphase is achieved by
UV-irradiation of the solution at 254 .mu.m using a xenon lamp, or
by addition of a radical initiator.
The resulting product contains microspheres with gas entrapped. The
microspheres are separated from excess polymerised amphiphiles
using a separating funnel. The resulting microspheres are
resuspended in sterile, pyrogen-free saline, and filled into 10 ml
vials. The product is produced using aseptic techniques in a "clean
room" (LAF-station) to obtain a sterile, pyrogen free product. The
particle sizes of the microspheres are in the range of 0.5-10
.mu.m.
EXAMPLE 2
Example 1 is repeated using as polymerisable amphiphile the
compound bis-(trieicoso-10,12-diynoyl) phosphatidyl choline (Hirth
et al; Helv Chim Acta 40, 957, 1928).
EXAMPLE 3
100 mg of bis-linoleyl-lecithin are dissolved in a mixture of
chloroform/methanol. The mixture is poured into a round bottom
flask, and the organic phase is evaporated using a rotavapor in
such a way that a thin film of the lecithin derivative is formed at
the inner surface of the flask. 10 ml of sterile, pyrogen-free-free
water are added and the lipids are dispersed in the solution by
sonication at the air/liquid interphase for 5-15 minutes. Gas
entrapped vesicles are formed, and the gas-containing microspheres
are polymerised by UV-irradiation of the solution at 254 nm using a
xenon-lamp or by addition of a radical initiator under continuous
stirring. Polymerised gas-entrapped vesicles are separated from
excess polymerised amphiphiles using a separating funnel. The
resulting vesicles are suspended in sterile, pyrogen free saline
and filtered to obtain a product which contains microspheres in the
range of 0.5-5 .mu.m. The product is produced using aseptic
techniques in a "clean room" (LAF-station) to obtain a sterile,
pyrogen free product. The final product is filled into 10 ml
vials.
EXAMPLE 4
Example 3 is repeated using as polymerisable amphiphile the
compound bis-(trieicoso-10,12-diynoyl) phosphatidyl choline (Hirth
et al; Helv Chim Acta 40, 957, 1928).
PREPARATION OF POLYMERISABLE AMPHIPHILES
EXAMPLE 5
Tetraethylene glycol mono-12-(methacryloyloxy)dodecanoate
12-(Methacryloyloxy)dodecanoic acid (Regen et. al., J. Am. Chem.
Soc. 1982, 104, 795) (2.75 g, 9.65 mmol) was dissolved in
tetrahydrofuran (45 ml) and a solution of oxalyl chloride (2.1 ml,
24.2 mmol) in tetrahydrofuran (5 ml) was added dropwise. The
mixture was stirred for 24 hours at room temperature, and then the
solvent was evaporated under reduced pressure. The residue was
dissolved in tetrahydrofuran (25 ml) and added dropwise to a
solution of tetraethylene glycol (1.88 g, 9.65 mmol) and pyridine
(0.92 g, 11.7 mmol) in tetrahydrofuran (35 ml). The mixture was
stirred for 24 hours at room temperature. The precipitated
pyridinium salt was filtered off and the solvent evaporated.
Chromatographic purification on a silica gel column (ethyl acetate)
afforded 1.67 g (38%) of the title compound. .sup.1 H NMR (60 MHz,
CDCl.sub.3), .delta. 1.3 (br s, 18H, (CH.sub.2).sub.9), 1.95 (m,
3H, C--CCH.sub.3), 2.1-2.6 (m , 2H, CH.sub.2 COO), 3.5-3.8 (m, 14H,
3.times.CH.sub.3 OCH.sub.3 CH.sub.3 +COOCH.sub.2 CH.sub.2), 4.0-4.4
(m, 4H, COOCH.sub.2), 5.52 (m, 1H, vinyl), 6.10 (m, 1H, vinyl).
EXAMPLE 6
Polyethylene glycol (550) methyl ether
12-(methacryloyloxy)dodecanoate
12-(Methacryloyloxy)dodecanoic acid (1.90 g, 6.69 mmol) was
dissolved in tetrahydrofuran (20 ml) and a solution of oxalyl
chloride (2.12 g, 16.7 mmol) in tetrahydrofuran (10 ml) was added
dropwise. The mixture was stirred for 24 hours at room temperature,
and then the solvent was evaporated under reduced pressure. The
residue was dissolved in tetrahydrofuran (10 ml) and added dropwise
to a solution of polyethylene glycol (550) monomethyl ether (3.68
g, 6.69 mmol) and pyridine (0.53 g, 6.69 mmol) in tetrahydrofuran
(25 ml). The mixture was stirred for 24 hours at room temperature.
The precipitated pyridinium salt was filtered off and the solvent
evaporated. Chromatographic purification on a silica gel column
(chloroform) afforded 2.31 g (42.3%) of the title compound. .sup.3
H NMR (60 MHz, CDCl.sub.3): .delta. 1.3 (br s, 18H,
(CH.sub.2).sub.9), 1.95 (m, 3H, C.dbd.CCH.sub.3), 2.1-2.5 (m, 2H,
CH.sub.2 COO), 3.11 (s, 3H, CH.sub.3 O), 3.5-3.8 (m, 25H (average),
CH.sub.2 OCH.sub.2 CH.sub.2 +COOCH.sub.2 CH.sub.2), 3.9-4.4 (m, 4H,
COOCH.sub.2), 5.52 (m, 1H, vinyl), 6.10 (m, 1H, vinyl).
EXAMPLE 7
Polyethylene glycol (2000) methyl ether
12-(methacryloyloxy)dodecanoate
12-(Methyacryloyloxy)dodecanoic acid (2184 g, 0.01 mol) in
tetrahydrofuran (20 ml) was reacted with oxalyl chloride (3.0 g,
0.024 mol) to obtain the corresponding acid chloride. This acid
chloride (3.0 g, 0.01 mol) dissolved in anhydrous tetrahydrofuran
(10 ml) was added dropwise to a mixture of polyethylene glycol
(2000) monomethyl ether (20.0 g, 0.01 mol) and anhydrous pryidine
(0.83 g, 0.01 mol) in anhydrous tetrahydrofuran (300 ml). The
mixture was stirred for 48 hours at room temperature. The resulting
liquid was purified by flash chromatography (silica gel/ethyl
acetate) to give 16.5 g (75%) of the title compound. .sup.1 H NMR
(60 MHz, CDCl.sub.3), .delta. 1.20 (s, 18H, CH.sub.2), 1.95 (m, 3H,
C.dbd.CH.sub.3), 2.15 (m, 2H, CH.sub.2 COO), 3.5 (s, 3H, CH.sub.3
O), 3.6 (s, 180H, 90.times.CH.sub.2), 4.0 (m, 4H,
2.times.COOCH.sub.2), 5.7-6.0 (m, 2H, CH.sub.2 .dbd.).
EXAMPLE 8
a) 16-(Methacryloyloxy)hexadecanoic acid
16-Hydroxyhexadecanoic acid (6.81 g, 25.0 mmol) was dissolved in
tetrahydrofuran (150 ml) and the solution was cooled to 0.degree.
C. before adding pyridine (2.73 g, 34.5 mmol). Methacryloyl
chloride (2.61 g, 25.0 mmol) was dissolved in tetrahydrofuran (75
ml) and added dropwise. The mixture was stirred for 1 hour at
0.degree. C., and then at room temperature for 24 hours. The
solvent was removed under reduced pressure (room temperature), the
residue suspended in either (100 ml) and the mixture washed with
distilled water. The ether layer was dried (MgSO.sub.4) and the
ether evaporated. Chromatographic purification on a silica gel
column (1:2 ethyl acetate/hexane) afforded 5.0 g (64%) of the title
compound. .sup.1 H NMR (60 MHz, CDCl.sub.3), .delta. 1.3 (br s,
26H, (CH.sub.2).sub.13), 1.95 (m, 3H, C.dbd.CCH.sub.3), 2.1-2.6 (m,
2H, CH.sub.2 COO ), 4.9-4.4 (m, 2H, COOCH.sub.2), 5.52 (m, 1H,
vinyl), 6.10 (M, 1H, vinyl).
b) Tetraethylene glycol mono-16-(methacryloyloxy)hexadecanoate
16-(methacryloyloxy)hexadecanoate
16-(Methacryloyloxy)hexadecanoic acid (2.05 g, 6.57 mmol) was
dissolved in tetrahydrofuran (25ml) in a solution of oxalyl
chloride (1.4 ml), 16.5 mmol) in tetrahydrofuran (10 ml) was added
dropwise. The mixture was stirred for 24 hours at room temperature,
and then the solvent was evaporated under reduced pressure. The
residue was dissolved in tetrahydrofuran (10 ml) and added dropwise
to a solution of tetraethylene glycol (1.07 g, 5.50 mmol) and
pyridine (0.44 g, 5.50 mmol) in tetrahydrofuran (25 ml). The
mixture was stirred for 24 hours at room temperature. The
precipitated pyridinium salt was filtered off and the solvent
evaporated. Chromatographic purification on a silica gel column
(2:1 ethyl acetate/hexane) afforded 0.84 g (30%) of the title
compound .sup.1 H NMR (60 MHz, CDCl.sub.3): .delta. 1.3 (br s, 26H,
(CH.sub.2).sub.13), 1.95 (m, 3H, C.dbd.CCH.sub.3), 2.1-2.6 (m, 2H,
CH.sub.2 COO), 3.5-3.8 (m, 14H, 3.times.CH.sub.2 OCH.sub.2 CH.sub.2
+COOCH.sub.2 CH.sub.2), 4.9-4.4 (m, 4H, COOCH.sub.2), 5.52 (m, 1H,
vinyl), 6.10 (m, 1H, vinyl).
EXAMPLE 9
Polyethylene glycol (350) methyl ether
16-(methacryloyloxy)hexadecanoate
The product was prepared from 16-(methacryloyloxy)-hexadecanoic
acid (prepared as described in Example 8(a)), and polyethylene
glycol (350) monomethyl ether using the procedure given in Example
6.
EXAMPLE 10
a) 12-(Acryloyloxy)dodecanoic acid
12-Hydroxydodecanoic acid (5.0 g, 0.023 mol) dissolved in
tetrahydrofuran (100 ml) and pyridine (2.16 g, 0.027 mol) was
cooled to 0.degree. C. Acryloyl chloride (3.15 g, 0.023 mol) in
tetrahydrofuran (75 ml) was then added dropwise to the solution.
The mixture was stirred for 5 hours at 0.degree. C. then stirred
overnight at room temperature. The precipitated pyridinium salt was
filtered off and the solvent removed under vacuum. The resulting
liquid was purified by flash chromatography (silica gel/chloroform)
to give 2.5 g (40%) of the title compound. .sup.1 H NMR (60 MHz,
CDCl.sub.3): .delta. 1.20 (s, 18H, CH.sub.2), 2.15 (m, 2H, CH.sub.2
COOH), 4.0 (m, 2H, COOCH.sub.2), 5.7-6.0 (m, 3H, CH.sub.2 .dbd. and
.dbd.CH).
b) Tetraethylene glycol mono- 12-(acryloyloxy) dodecanoate
12-Acryloyloxydodecanoic acid (2.00 g, 0.007 mol) in diethyl ether
(20 ml) was reacted with oxalyl chloride (2.40 g, 0.019 mol) to
obtain the corresponding acid chloride. This acid chloride (1.80 g,
0.006 mol) dissolved in anhydrous chloroform (10 ml) was added
dropwise to a mixture of tetraethylene glycol (1.20 g, 0.006 mol)
and anhydrous pyridine (0.50 g, 0.006 mol) in anhydrous chloroform
(30 ml). The mixture was stirred overnight at room temperature. The
resulting liquid was purified by flash chromatography (silica
gel/ethyl acetate) to give 1.10 g (40%) of the title compound as a
colourless oil. .sup.1 H NMR (60 MHz, CDCl.sub.3): .delta. 1.20 (s,
18H, CH.sub.2), 2.15 (m, 2H, CH.sub.2 COOH), 3.50 (s, 3H, CH.sub.3
O), 3.6 (s, 14H, 7.times.CH.sub.2 O), 4.0 (m, 5H,
2.times.COOCH.sub.3 and OH), 5.7-6.0 (m, 3H, CH.sub.2 .dbd. and
.dbd.CH).
EXAMPLE 11
Tetraethylene glycol mono- 10,12-tricosadiynoate
10,12-Tricosadiynoic acid (2.50 g, 0.007 mol) in tetrahydrofuran
(30 ml) was reacted with oxalyl chloride (2.25 g, 0.017 mol) to
obtain the corresponding acid chloride. This acid chloride (2.45 g,
0.007 mol) dissolved in anhydrous tetrahydrofuran (10 ml) was added
dropwise to a mixture of tetraethylene glycol (1.32 g, 0.007 mol)
and anhydrous pyridine (0.83 g, 0.01 mol) in anhydrous
tetrahydrofuran (40 ml). The mixture was stirred overnight at room
temperature. The precipitated pyridinium salt was filtered off and
the solvent removed under vacuum. The resulting liquid was purified
by flash chromatography (silica gel/ethyl acetate) to give 1.50 g
(41%) of the title compound as a colour less oil. .sup.1 H NMR (60
MHz, CDCl.sub.3): .delta. 0.88 (m, 3H, CH.sub.3 CH.sub.2), 1.30 (m,
28H, CH.sub.2), 2.20 (m, 6H, CH.sub.2), 3.65 (s, 11H,
7.times.CH.sub.2 O), 4.20 (m, 2H, CH.sub.2 CO).
EXAMPLE 12
Polyethylene glycol (550) methyl ether 10,12-tricosadiynoate
10,12-Tricosadiynoic acid (2.50 g, 0.007 mol) in tetrahydrofuran
(30 ml) was reacted with oxalyl chloride (2.25 g, 0.017 mol) to
obtain the corresponding acid chloride. This acid chloride (2.45 g,
0.007 mol) dissolved in anhydrous tetrahydrofuran (10 ml) was added
dropwise to a mixture of polyethylene glycol (550 ) monomethyl
ether (3.85 g, 0.007 mol) and anhydrous pyridine (0.83 g, 0.01 mol)
in anhydrous tetrahydrofuran (30 ml). The mixture was stirred
overnight at room temperature. The precipitated pyridinium salt was
filtered off and the solvent removed under vacuum. The resulting
liquid was purified by flash chromatography (silica gel/ethyl
acetate) to give 2.72 g (41%) of the title compound as a colourless
oil. .sup.1 H NMR (60 MHz, CDCl.sub.3): .delta. 0.88 (m, 3H,
CH.sub.3 CH.sub.2), 1.30 (m, 28H, CH.sub.2), 2.20 (m, 6H,
CH.sub.2), 3.65 (s, 48H, 24.times.CH.sub.2 CO), 3.50 (s, 3H,
CH.sub.3 O), 4.20 (m, 2H, CH.sub.2 CO).
EXAMPLE 13
a) Methyl 10,12-tricosadiynoate
10,12- Tricosadiynoic acid (3.0 g, 0.0084 mol), methanol (15 ml)
and concentrated sulfuric acid (0.8 ml) were heated to reflux and
stirred for 1 hour. The cooled mixture was taken up in either (40
ml) and washed with 10% NaHCO.sub.3 (20 ml) and water (20 ml), and
the organic phase was dried (MgSO.sub.4). Evaporation of the
solvent gave 2.68 g (74%) of the title compound. .sup.1 H NMR (60
MHz, CDCl.sub.3): .delta. 0.98 (m, 3H, CH.sub.3 CH.sub.2), 1.28 (m,
28H, CH.sub.2), 2.25 (m, 6H, CH.sub.2), 3.70 (s, 3H, CH.sub.2
O).
b) N-(2',3'-Dihydroxypropyl)-10,12-tricosadiynamide
methyl 10,12-tricosadiynoate (1.69 g, 4.67 mmol) was dissolved in
methanol. 3-Amino-1,2-propanediol (0.509 g, 5.6 mmol) and sodium
methoxide 2.5% solution in methanol (0.146 g, 3 mol %) was added.
The mixture was refluxed for 3 hours and the solvent evaporated.
The crude product was recrystallized from chloroform. Yield: 1.00 g
(51%). .sup.1 H NMR (60 MHz, CDCl.sub.3): .delta. 0.7-1.0 (m, 3H,
CH.sub.3 CH.sub.2), 1.3 (s, br, 28H, CH.sub.2), 2.0-2.4 (m, 6H,
CH.sub.2), 3.3-3.8 (m, 5H, 2.times.CH.sub.2 +CH (propanediol)),
6.0-6.3 (m, 1H, NH).
EXAMPLE 14
N,N'-bis(2,3-dihydroxypropyl)2,4,6-triiodo-5-(tricosa-10,12-diynoylamino)is
ophthalamide
5-Amino, N,N'-bis(2,3-diacetoxypropyl)-2,4,6-triiodoisophthalamide
(2.19 g, 2.5 mmol) and 10,12-tricosadiynoyl chloride (1.82 g, 5
mmol) were dissolved in 20 ml dichloromethane. The solution was
stirred for 3 days at ambient temperature under a nitrogen
atmosphere. TLC (ethyl acetate) indicated that the reaction was
complete. The reaction mixture was evaporated and dissolved in a
mixture of methanol (30 ml) and 1M sodium hydroxide solution (15
ml). After 1 hour TLC (methanol/chloroform) indicated that the
reaction was complete. The solution was neutralized with
concentrated hydrochloric acid. The residue was dissolved in
chloroform and filtered. The solvent was removed and the reaction
mixture was purified through silica gel with methanol/chloroform
(1:3) to give the title compound. .sup.1 H NMR (300 MHz, DMSO):
.delta. 0.8 (CH.sub.3, t), 1.2-1.7 (17.times.CH.sub.2, m), 2.2-2.3
(2.times.CH.sub.2, t), 3.1-3.2 (2.times.CH.sub.2 NH, m), 3.3-3.5
(2.times.CH.sub.2 OH, m), 3.6-3.8 (2.times.CHOH), 4.4-4.7
(4.times.OH), m), 8.4-8.5 (2.times.CONH, m), 9.8 (2.times.ArNHCO,
s).
EXAMPLE 15
N-(3',4',5'-Trihydroxy-6'-hydroxymethyltetrahydropyran-2'-yl)-10,12-tricosa
diynamide
1-Amino-1-deoxy -.beta.-D-galactose (180 mg, 1 mmol),
10,12-tricosadiynoic acid (350 mg, 1 mmol) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were dissolved in 25
ml dry dimethylformamide and stirred at room temperature overnight.
The solvent was removed in vacuo, the residue redissolved in
chloroform/methanol (1:1), filtered and purified by straight phase
chromatography on a CHROMATOTRON. The relevant fractions were
collected, concentrated in vacuo, and the product was characterised
by NMR.
EXAMPLE 16
6-(2',6'-(Diaminohexanoylamino)-3,4,5-trihydroxytetra-hydropyran-2-ylmethyl
10,12-tricosadiynoate
1-Amino-1-deoxy-.beta.-D-galactose (180 mg, 1 mmol), and
Fmoc-Lys(Boc)-OPfp (650 mg, 1 mmol) were dissolved in 4 ml dry
dimethylformamide and stirred at room temperature overnight. The
solvent was removed in vacuo, the residue was redissolved in
acetonitrile/water (1:1), filtered and purified by reversed phase
chromatography (Lobar RP8B, acetonitrile/water 50:50 and 65:35).
The relevant fractions were collected, concentrated in vacuo, and
the product was characterised by NMR. The purified product (1 g, 1
mmol), 10,12-tricosadiynoic acid (350 mg, 1 mmol) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are dissolved in 10
ml dry dimethylformamide and stirred at room temperature overnight.
The solvent is removed in vacuo, the residue redissolved in
chloroform/methanol (95:5), filtered and purified by straight phase
chromatography on a CHROMATOTRON. The relevant fraction are
collected, concentrated in vacuo, and the product is characterised
by NMR. The protecting groups of the .alpha.-.epsilon. amino groups
are removed by standard reactions. Boc is removed by treatment with
trifluoroacetic acid/methylene chloride for 30 minutes. The solvent
is removed in vacuo. Fmoc is removed by treating the residue with
20% piperidine in dimethylformamide for 30 minutes, and the solvent
is removed in vacuo. The final product is purified by reversed
phase chromatography (Labor RP8B).
EXAMPLE 17
(3,4,5,6-Tetrahydroxytetrahydropyran-2-ylmethyl)
10,12-tricosadiynoate
1,2,3,4-di-O-isopropylidene-D-galactopyranose (2.6 g, 10 mmol) and
10,12-tricosadiynoic acid (3.5 g, 10 mmol) were dissolved in 25 ml
methylene chloride. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(2 g, >10 mmol) was added neat. The reaction mixture was stirred
overnight at room temperature. The reaction mixture was diluted to
100 ml, extracted with water (2.times.25 ml), dried over MgSO.sub.4
and the solvent was remove din vacuo. The crude product was treated
with trifluoroacetic acid (10 ml) at room temperature for 30
minutes, evaporated in vacuo, and purified by straight phase
chromatography on a CHROMATOTRON, eluted with methanol/chloroform
(5:95). The product was characterised by NMR.
PREPARATION OF ULTRASOUND CONTRAST AGENTS
EXAMPLES 18-41
i) General preparative procedure
The polymerisable amphiphile was dissolved in a minimum of methanol
and added to a mixture of water and a hydrocarbon. A comonomer
and/or 2,2'-azobisisobutyronitrile (AIBN) dissolved in a minimum of
methylene chloride were optionally added and nitrogen was bubbled
through the mixture for 1 minute, whereafter the mixture was
sonicated under a nitrogen atmosphere using a LABSONIC 2000
apparatus, the sonication probe (length 127 mm, diameter 9.5 mm)
being placed 2-3 cm below the surface of the mixture and the energy
used being "full scale" or "half scale" in the low position. The
resulting emulsions were optionally irradiated with UV light under
a nitrogen atmosphere or treated with a redox initiator comprising
potassium metabisulphite (0.05 g, 0.22 mmol) in water (1 ml) and
potassium peroxosulphate (0.0023 g, 3.3.times.10.sup.-3 mmol) in
water (1 ml). The procedure was modified in Example 31 in that AIBN
was added and the mixture was then shaken by hand, whereafter a
first portion of comonomer was added and sonication was effected
while nitrogen gas was bubbled through the mixture. A further
portion of comonomer was then added and the resulting emulsion
subjected to UV irradiation.
The specific reaction conditions employed in each Example are set
out in Table 1. Similar conditions, e.g. involving sonication for 5
minutes using the full scale setting and irradiating for 1 hour or
adding the above-described redox initiator system and stirring
carefully for 30 minutes, may be employed to treat the amphiphiles
prepared in Examples 14-17.
__________________________________________________________________________
Example in which amphiphile prepared Volume Hydrocarbon Comonomer
Quantity Sonication level Duration of Example and quantity used of
water and volume and quantity of AIBN and duration UV irradiation
Redox No (g/mmol) (ml) (ml) (g/mmol) (g/mmol) (minutes) (hours)
system
__________________________________________________________________________
5 MM 16 0.039/0.084 50 PE-5 0.018/0.18 -- fs-5 -- -- 5 MM 19
0.037/0.080 50 IP-5 0.018/0.18 -- hs-3 -- -- 5 MM 20 0.383/0.83 500
IP-50 0.18/1.8 0.20/1.21 fs-6 1.5 -- 5 MM 21 0.042/0.091 50 IP-5
0.09/0.9 0.02/0.12 fs-3 1 -- 5 MM 22 0.040/0.086 50 PE-2.5
0.018/0.18 0.02/0.12 fs-3 1 -- 6 MM 23 0.053/0.065 50 PE-5
0.018/0.18 0.02/0.12 fs-4 -- -- 6 MM 24 0.530/0.65 500 PE-50
0.180/1.80 0.200/1.20 fs-8 1 -- 6 MM 25 0.500/0.61 500 PE-50
0.180/1.80 0.200/1.20 fs-8 2.5 -- 6 MM 26 0.200/0.245 20 PE-2
0.018/0.18 0.020/0.12 fs-3 1 .check mark. (Ex 26a) (Ex 26b) 6 MM 27
0.053/0.063 50 PE-25 0.018/0.18 0.020/0.12 fs-3 -- .check mark. 6
MM 28 0.054/0.066 50 PE-1 0.018/0.18 0.020/0.12 fs-3 -- -- 6 MM 29
0.054/0.066 50 TO-5 0.018/0.18 0.020/0.12 fs-3 -- --
6 ST 30 0.056/0.069 50 PE-5 0.042/0.41 0.020/0.12 fs-3 1 -- 6 ST 31
0.057/0.070 50 PE-5 0.042/0.41 + 0.020/0.12 fs-3 1 -- 0.099/0.95 6
32 0.054/0.066 50 IP-5 -- 0.020/0.12 fs-6 -- -- 7 MM 33 0.193/0.090
50 PE-5 0.018/0.18 0.02/0.12 fs-3 1 -- 8(b) MM 34 0.042/0.081 50
IP-5 0.018/0.18 0.020/0.12 hs-3 1 -- 8(b) ST 35 0.046/0.089 50 PE-5
0.042/0.41 0.020/0.12 fs-3 9 36 0.052/0.077 50 PE-5 -- -- fs-3 --
-- 10(b) MM 37 0.036/0.08 50 PE-5 0.018/0.18 0.02/0.12 fs-3 1 -- 11
38 0.047/0.09 50 PE-5 -- 0.02/0.12 fs-6 -- -- 11 39 0.050/0.15 50
PE-5 -- 0.02/0.12 fs-3 1 -- 12 40 0.057/0.06 50 PE-5 -- 0.02/0.12
fs-3 -- .check mark." 13(o) 41 0.046/0.11 50 PE-5 -- 0.02/0.12 fs-3
-- --
__________________________________________________________________________
KEY PE = petroleumether (b.p. 40-60.degree. C.): IP = isopentane;
TO = toluene; MM = methylmethacrylate; ST = styrene; fs = full
scale; hs = hal scale "Amount of potassium peroxosulphate reduced
to 0.002 g (0.003 mmol)
ii) Acoustic characterisation
The acoustic effects of the products of Examples 18-41 were
investigated by measuring their ultrasonic transmission as a
function of time, over a period of 90 seconds. The tests were
performed on samples of emulsified material as formed immediately
after sonication and, where appropriate, on the material after
subjection to UV irradiation or redox initiation. In the case of
Example 25 the sample removed after irradiation was retreated after
dilution with water (1:1). In the case of Example 31 a sample
removed after the manual shaking was also tested. A 3.5 MHz
broadband transducer was used in a pulse-reflection technique. All
the readings were stable during the 90 seconds measurement period,
so that a single value (in dB/cm) is sufficient to describe each 90
second measurement. In certain cases the measurements were repeated
at time intervals to investigate further the stability of the
ultrasound contrast agents. The results are presented in Table 2,
the time intervals (in minutes from sonication) to acoustic
characterisation are given in brackets for each reading.
TABLE 2 ______________________________________ Acoustic
characterisations Acoustic effect after UV Acoustic effect
irradiation/redox Example No. after sonication initiation
______________________________________ 18 2.6 (0) 19 3.7 (0) 20 3.7
(0) 1.4 (90) 1.7 (90) 21 0.6 (0) 0 (60) 22 0.7 (0) 0.5 (60) 0.9 (5)
0 (120) 23 5.9 (0) 4.3 (104) 24 6.0 (0) 4.1 (60) 25 4.4 (0) 4.2
(30) 2.9 (150) 2.9 (150) 1.4 (150) diluted 26 4.0 (0) 2.8 (20,
redox) 1.8 60) 0.4 (60, UV) 27 3.6 (0) 2.9 (10) 3.2 (10) 2.3 (60)
3.6 (60) 0.6 (720) 28 0.9 (0) 29 0.6 (0) 30 5.7 (0) 4.1 (60) 3.2
(60) 3.2 (150) 2.6 (150) 31 2.5 (after shaking) 5.4 (0) 4.0 (60)
2.2 (60) 3.3 (150) 1.7 (150) 32 4.9 (0) 33 5.5 (0) 4.7 (20) 3.5
(60) 2.4 (60) 3.1 (100) 34 2.2 (0) 0 (60) 35 1.1 (0) 36 2.1 (0) 37
1.7 (0) 0 (60) 38 4.5 (0) 39 5.6 (0) 4.7 (60) 4.9 (60) 4.5 (120)
4.3 (120) 40 3.6 (0) 0 (60) 41 5.3 (0)
______________________________________
iii) Microscopy analysis
A selection of the products from Examples 18-41 were investigated
using a light microscope (Nikon UFX-II) with a micrometer scale.
The investigations were generally performed by taking out samples
of emulsified material as formed immediately after sonication,
except for Example 31 (where the sample was withdrawn after manual
shaking), Example 39 (where the sample was withdrawn after UV
irradiation) and Example 40 (where samples were withdrawn both
after sonication and after redox initiation), and placing each
sample between two glass plates. The results of these
investigations are presented in Table 3; the time intervals (in
minutes from sonication) to microscopy analysis are given for each
sample.
TABLE 3 ______________________________________ Microscopy analysis
Time after Comments (shape, sonication Size size Example No. (min)
(diam., .mu.m) distribution) ______________________________________
25 10 4 spheres, narrow size distribution 26 10 10-25 spheres 27 10
4 spheres, narrow size distribution 28 10 4-6 spheres, narrow size
distribution 29 10 variable various shapes, broad size distribution
30 10 4-6 spheres, narrow size distribution 31 10 (after 10-100
large bubbles, shaking) unlike the sonicated samples 33 10 2-3
spheres 35 10 10-15 spheres 36 10 8-15 spheres, broad size
distribution 38 10 5-10 spheres 39 10 5-10 spheres, also larger
bubbles 40 30 5-10 spheres 40 30 (after variable bubbles of redox)
irregular shape 41 10 4 spheres, narrow size distribution
______________________________________
iv) Size Exclusion Chromatography
Size Exclusion Chromatrography (SEC) was performed on the freeze
dried product from Example 25 using tetrahydrofuran (Rathburn HPLC
quality) as eluant and refractive index as detector (Knauer,
Germany). The column set used consisted of 3.times.30 cm columns
containing 5 .mu.m styrogel with pore sizes of 10.sup.5, 10.sup.4,
and 500 .ANG. (Polymer Laboratories Ltd., England). Calibration was
made against polystyrene standards (Polymer Laboratories Ltd.,
England). The amphiphilic monomer starting material gave a peak
molecular weight of 1,600 Daltons and the polymer product gave a
peak molecular weight of 22,000 Daltons, both given in polystyrene
equivalents. Using the conversion factor of 0.59 for converting
from polystyrene equivalents to "real" molecular weights (the value
for PEG given by Dawkins et al., J. Liq. Chromatog. 7, 1739,
(1984)), these correspond to molecular weights of 944 Daltons for
the monomer and 13,000 Daltons for the polymer respectively.
EXAMPLES 42-49
General Procedure
1,2-Dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) was
dissolved in a solution of 50 mg of propylene glycol/glycerol
(3:10) in 1 ml water to a final concentration of 5 mg
phospholipid/ml solution. 0.8 ml portions of this stock solution
were transferred to 2 ml vials with screw caps, whereafter the head
space was flushed with perfluorobutane gas. The vials were
vigorously shaken for 45 seconds, and transferred to a table roller
for approximately 30 minutes. Stock solutions of
crosslinkers/polymerisation activating agents were prepared by
dissolving the reagent in water to a concentration where 0.1 ml
solution contained 1 equivalent of glutaraldehyde or 2 equivalents
of N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC). The reagent solution was added via a pipette tip to the
bottom of each vial in a single application, whereafter the
headspace was flushed with perfluorobutane gas, and the vial was
transferred to the table roller and rolled for approximately 3
hours. The vials were centrifuged for 5 minutes at 2000 rpm at
20.degree. C., whereafter the infranatant was removed from the
bottom of the vial by a syringe and substituted with an equivalent
volume of degassed water. The head space was flushed with
perfluorobutane gas, and rolling was continued until a homogeneous
dispersion was obtained. This washing procedure was repeated
twice.
EXAMPLE 42
The phospholipid was reacted with 0.1 ml glutaraldehyde solution.
The product was characterized by Coulter counter analysis (number
and size distribution) and in vitro echogenicity measurements, and
was found to be stable at room temperature for several days.
EXAMPLE 43
The phospholipid was reacted with 0.1 ml EDC solution. The product
was characterised by Coulter counter analysis (number and size
distribution) and in vitro echogenicity measurements, and was found
to be stable at room temperature for several days.
EXAMPLE 44
The phospholipid was prewashed for 30 minutes on the table roller,
whereafter the vials were centrifuged for 5 minutes at 2000 rpm at
20.degree. C., the infranatant was removed from the bottom of the
vial by a syringe and substituted with an equivalent volume of
degassed water. The headspace was flushed with perfluorobutane gas
and rolling was continued until a homogeneous dispersion was
obtained. This prewashed phospholipid was then reacted with 0.1 ml
glutaraldehyde solution. The product was characterised by Coulter
counter analysis (number and size distribution) and in vitro
echogenicity measurements, and was found to be stable at room
temperature for several days.
EXAMPLE 45
Phospholipid prewashed as in Example 44 was reacted with 0.1 ml EDC
solution. The product was characterised by Coulter counter analysis
(number and size distribution) and in vitro echogenicity
measurements, and was found to be stable at room temperature for
several days.
EXAMPLE 46
The phospholipid was reacted with 0.1 ml glutaraldehyde solution.
The resulting suspension was frozen and lyophilised.
EXAMPLE 47
The phospholipid was reacted with 0.1 ml EDC solution. The
resulting suspension was frozen and lyophilised.
EXAMPLE 48
The phospholipid was reacted with 0.1 ml glutaraldehyde solution.
In the final washing step the infranatant was substituted with a
20% solution of glucose as cryoprotectant. The resulting suspension
was frozen and lyophilised.
EXAMPLE 49
The phospholipid was reacted with 0.1 ml EDC solution. In the final
washing step the infranatant was substituted with a 20% solution of
glucose as cryoprotectant. The resulting suspension was frozen and
lyophilised.
EXAMPLE 50
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt)
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (5.0 mg in distilled water (1 ml) in a 2 ml vial with
a septum was vigorously shaken for 15 seconds, heated to 60.degree.
C. for 10 minutes and then cooled to 20.degree. C. Perfluoropentane
(1.2 .mu.l) was added and the vial was vigorously shaken for 30
seconds to give a suspension of perfluoropentane/air microbubbles
of size 2-4 .mu.m as determined by light microscopy. This
suspension was stable for several days at room temperature.
b) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous suspension from (a) above. The vial was placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 2-4 .mu.m. The suspension was
stale for several days at room temperature.
c) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25%, 16.5 mg) was added to
the aqueous suspension from (a) above. The vial was placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 2-4 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 51
Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt)
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (4.6 mg) in distilled water (1 ml) in a 2 ml vial
with a septum was vigorously shaken for 15 seconds, heated to
60.degree. C. for 10 minutes and then cooled to 20.degree. C.
Perfluoropentane (2.4 .mu.l) was added and the vial was vigorously
shaken for 30 seconds to give a suspension of perfluoropentane/air
microbubbles of size 2-5 .mu.m as determined by light microscopy.
This suspension was stable for several days at room
temperature.
EXAMPLE 52
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt)
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (5.2 mg) in distilled water (1 ml) in a 2 ml vial
with a septum was vigorously shaken for 15 seconds, heated to
60.degree. C. for 10 minutes and then cooled to 20.degree. C.
Perfluoropentane (5 mg) was added and the vial was vigorously
shaken for 30 seconds to give a suspension of perfluoropentane/air
microbubbles of size 2-5 .mu.m as determined by light microscopy.
This suspension was stable for several days at room
temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25%, 16.5 mg) was added to
the aqueous suspension from (a) above. The vial was placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 2-4 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 53
a) Perfluorobutane/perfluorohexane microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt)
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (5.0 mg) in distilled water (1 ml) in a 2 ml vial
with a septum was vigorously shaken for 15 seconds, heated to
60.degree. C. for 10 minutes and then cooled to 20.degree. C. The
vial was evacuated at 10 mm Hg for 20 minutes to remove air
whereafter the headspace was flushed with perfluorobutane.
Perfluorohexane (1.4 .mu.l) was added and the vial was vigorously
shaken for 30 seconds to give a suspension of
perfluorobutane/perfluorohexane microbubbles of size 1-10 .mu.m as
determined by light microscopy. This suspension was stable for
several days at room temperature.
b) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous solution from (a) above. The vial was placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 2-4 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 54
Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt)
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (3.0 mg) and propylene glycol/glycerol (3:10, 46 mg)
in distilled water (1 ml) in a 2 ml vial with a septum was
vigorously shaken for 1 minute. Perfluoropentane (4.5 .mu.l) was
added and the vial was vigorously shaken for 30 seconds to give a
suspension of perfluoropentane/air microbubbles of size 2-10 .mu.m
as determined by light microscopy. This suspension was stable for
several hours at room temperature.
b) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous suspension from (a) above and the resulting mixture
was vigorously shaken for 5 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-8 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 55
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (2.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (0.5 mg) in distilled
water (1 ml) was heated at 80.degree. C. for 1 hour.
Perfluoropentane (1.2 .mu.l) was then added and the mixture was
vigorously shaken for 30 seconds to give a suspension of
perfluoropentane/air microbubbles of size 1-8 .mu.m as determined
by light microscopy. This suspension was stable for several hours
at room temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25%, 16.5 mg) was added to
the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 5 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-5 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 56
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (4.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (0.5 mg) in distilled
water (1 ml) was heated at 80.degree. C. for 1 hour.
Perfluoropentane (1.2 .mu.l) was then added and the mixture was
vigorously shaken for 30 seconds to give a suspension of
perfluoropentane/air microbubbles of size 1-10 .mu.m as determined
by light microscopy. This suspension was stable for several hours
at room temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25%, 16.5 mg) was added to
the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-5 .mu.m. The suspension was
stable for several days at room temperature.
c) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-5 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 57
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (2.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (0.7 mg) in distilled
water (1 ml) was heated at 80.degree. C. for 1 hour.
Perfluoropentane (5 mg) was then added and the mixture was
vigorously shaken for 30 seconds to give a suspension of
perfluoropentane/air microbubbles of size 1-8 .mu.m as determined
by light microscopy. This suspension was stable for several hours
at room temperature.
b) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-5 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 58
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (2.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (0.6 mg) and propylene
glycol/glycerol (3:10, 40 mg) in distilled water (1 ml) was heated
at 80.degree. C. for 1 hour. Perfluoropentane (1.4 .mu.l) was then
added and the mixture was vigorously shaken for 30 seconds to give
a suspension of perfluoropentane/air microbubbles of size 1-10
.mu.m as determined by light microscopy. This suspension was stable
for several hours at room temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25%, 16.5 mg) was added to
the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-5 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 59
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (4.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1.0 mg) and propylene
glycol/glycerol (3:10, 45 mg) in distilled water (1 ml) was heated
at 80.degree. C. for 1 hour. Perfluoropentane (1.2 .mu.l) was added
and the mixture was vigorously shaken for 30 seconds to give a
suspension of perfluoropentane/air microbubbles of size 1-10 .mu.m
as determined by light microscopy. This suspension was stable for
several hours at room temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25%, 16.5 mg) was added to
the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-10 .mu.m. The suspension was
stable for several days at room temperature.
b) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-5 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 60
a) Perfluoropentane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (2.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (0.5 mg) and propylene
glycol/glycerol (3:10, 40 mg) in distilled water (1 ml) was heated
at 80.degree. C. for 1 hour. Perfluoropentane (2.5 .mu.l) was added
and the mixture was vigorously shaken for 30 seconds to give a
suspension of perfluoropentane/air microbubbles of size 1-10 .mu.m
as determined by light microscopy. This suspension was stable for
several hours at room temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25%, 16.5 mg) was added to
the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 5 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-7 .mu.m. The suspension was
stable for several days at room temperature.
c) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 5 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 1-5 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 61
a) Perfluorohexane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (2.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (0.5 mg) in distilled
water (1 ml) was heated at 80.degree. C. for 1 hour.
Perfluoropentane (1.4 .mu.l) was added and the mixture was
vigorously shaken for 30 seconds to give a suspension of
perfluorohexane/air microbubbles of size 2-10 .mu.m as determined
by light microscopy. This suspension was stable for several hours
at room temperature.
b) Polymerisation of the phospholipid
An aqueous solution of EDC (0.1 mg in 3 drops of water) was added
to the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel,
tilted by about 60.degree., for 20 hours at 20.degree. C. The size
of the resulting microbubbles, as estimated by light microscopy,
was 2-10 .mu.m. The suspension was stable for several days at room
temperature.
EXAMPLE 62
a) Perfluorohexane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (2.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1.3 mg) in distilled
water (1 ml) was heated at 80.degree. C. for 1 hour.
Perfluoropentane (1.4 .mu.l) was added and the mixture was
vigorously shaken for 30 seconds to give a suspension of
perfluorohexane/air microbubbles of size 2-10 .mu.m as determined
by light microscopy. This suspension was stable for several hours
at room temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25% 16.5 mg) was added to
the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 2-10 .mu.m. The suspension was
stable for several days at room temperature.
EXAMPLE 63
a) Perfluorohexane/air microbubbles prepared using
1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine) (sodium salt) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine
A solution of 1,2-dipalmitoyl-sn-glycero-3-(phospho-L-serine)
(sodium salt) (2.5 mg) and
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (0.6 mg) and propylene
glycol/glycerol (3:10, 45 mg) in distilled water (1 ml) was heated
at 80.degree. C. for 1 hour. Perfluorohexane (2.4 .mu.l) was added
and the mixture was vigorously shaken for 30 seconds to give a
suspension of perfluorohexane/air microbubbles of size 2-10 .mu.m
as determined by light microscopy. This suspension was stable for
several hours at room temperature.
b) Crosslinking of the phospholipid
An aqueous solution of glutaraldehyde (25% 16.5 mg) was added to
the aqueous suspension from (a) above, and the mixture was
vigorously shaken for 3 seconds. The vial was then placed in a
slowly rotating carousel, tilted by about 60.degree., for 20 hours
at 20.degree. C. The size of the resulting microbubbles, as
estimated by light microscopy, was 2-10 .mu.m. The suspension was
stable for several days at room temperature.
* * * * *